Differential TGF-β1/SMAD4 Signaling Regulates PMN-MDSC Differentiation and Reshapes the Immune Microenvironment in Breast Cancer

Jie Cao,1,2 Enqi Qiao,1,2 Hui Shao,2,3 Chunlei Wang,1,2 Manzhi Xia,1,2 Bo Qin4 1Department of General Surgery, Shaoxing Maternity and Child Health Care Hospital, Shaoxing, Zhejiang, People’s Republic of China; 2School of Medicine, Maternity and Child Health Care Affiliated Hospital, Shaoxing University, Shaoxing, Zhejiang, People’s Republic of China; 3Infection Control Office, Shaoxing Maternity and Child Health Care Hospital, Shaoxing, Zhejiang, People’s Republic of China; 4Department of Laboratory Medicine, Shaoxing Maternity and Child Health Care Hospital, Shaoxing, Zhejiang, People’s Republic of ChinaCorrespondence: Bo Qin, Department of Laboratory Medicine, Shaoxing Maternity and Child Health Care Hospital, Shaoxing, Zhejiang, People’s Republic of China, Email [email protected]: Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) exert strong immunosuppressive effects and are associated with poor prognosis in breast cancer. However, the mechanism underlying their differentiation remains unclear. Here, we aimed to elucidate how transforming growth factor-β 1 (TGF-β 1)/SMAD4 signaling regulates PMN-MDSC differentiation and modulates the breast cancer immune microenvironment.Methods: We analyzed neutrophil subpopulation heterogeneity in breast cancer tissues using single-cell RNA sequencing combined with spatial transcriptomics (GSE176078 dataset). A 4T1 mouse breast cancer metastasis model was established, and tumor progression was tracked via in vivo fluorescence imaging. Colony formation and differentiation outcomes following Tgfb1 overexpression or knockdown were assessed in spleen-derived hematopoietic stem cells using flow cytometry. Gene expression profiles and signaling pathways were characterized using RNA sequencing and Western blotting.Results: Neutrophil subpopulations in breast cancer tissues exhibited significant heterogeneity, particularly the N3 subset, which showed enhanced intracellular communication and regulatory potential. Tgfb1 was significantly upregulated along the neutrophil developmental trajectory in both spleen tissue and serum of breast cancer mice. In normal mice, the number of metastatic foci significantly correlated with neutrophil proportions. Tgfb1 overexpression promoted PMN-MDSC differentiation, colony formation, and SMAD4 upregulation in normal mice but exerted opposite effects in tumor-bearing mice.Conclusion: Our findings implicate TGF-β 1/SMAD4 signaling in the differential regulation of PMN-MDSC differentiation in breast cancer models. The TGF-β 1 pathway represents a potential therapeutic target for modulating the immune microenvironment in breast cancer, although further validation in clinical settings is required to determine therapeutic efficacy.Plain Language Summary: This study demonstrates that the transforming growth factor-β 1 (TGF-β 1)/SMAD4 signaling pathway differentially regulates the differentiation of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in breast cancer. Specifically, TGF-β 1 promotes the development of immunosuppressive cells under healthy conditions, whereas it exerts opposing effects in tumor-bearing environments. These findings suggest that TGF-β 1 represents a promising therapeutic target for modulating the immune microenvironment in breast cancer and enhancing clinical outcomes for patients. Keywords: immunotherapy, polymorphonuclear neutrophil, myeloid-derived suppressor cell, tumor microenvironment